The compounds of the invention are inhibitors of bacterial polypeptide deformylase (PDF; EC 3.5.1.31).
All ribosome-mediated synthesis of proteins starts with a methionine residue. In prokaryotes, the methionyl moiety carried by the initiator tRNA is N-formylated prior to its incorporation into a polypeptide. Consequently, N-formylmethionine is always present at the N-terminus of a nascent bacterial polypeptide. However, most mature proteins do not retain the N-formyl group or the terminal methionine residue. Deformylation is required prior to methionine removal, since methionine aminopeptidase does not recognise peptides with an N-terminal formylmethionine residue (Solbiati et al., J. Mol. Biol. 290: 607–614, 1999). Deformylation is, therefore, a crucial step in bacterial protein biosynthesis and the enzyme responsible, PDF, is essential for normal bacterial growth. The gene encoding PDF (def) is present in all pathogenic bacteria for which sequences are known (Meinnel et al., J. Mol. Biol, 266: 939–49, 1997). Although a deformylase homologue has recently been cloned from the mitochondria of human cells (Giglione et. el. EMBO Journal, 19, 5916–5929, 2000) it has not been shown to be functional, and its relevance is unknown. Since a number of currently used antibiotics are known to act on both bacteria and mitochondria, PDF is still considered to be a target for antibacterial chemotherapy (for a review see Giglione et al., Mol Microbiol., 36: 1197–1205, 2000).
The isolation and characterisation of PDF has been facilitated by an understanding of the importance of the metal ion in the active site (Groche et al., Biophys. Biochem. Res. Commun., 246: 324–6, 1998). The Fe2+ form is highly active in vivo but is unstable when isolated due to oxidative degradation (Rajagopalan et al., J. Biol. Chem. 273: 22305–10, 1998). The Ni2+ form of the enzyme has specific activity comparable with the ferrous enzyme but is oxygen-insensitive (Ragusa et al., J. Mol. Biol. 1998, 280: 515–23, 1998). The Zn2+ enzyme is also stable but is almost devoid of catalytic activity (Rajagopalan et al., J. Am. Chem. Soc. 119: 12418–12419, 1997).
Several X-ray crystal structures and NMR structures of E. coli PDF, with or without bound inhibitors, have been published (Chan et al., Biochemistry 36: 13904–9, 1997; Becker et al., Nature Struct. Biol. 5: 1053–8, 1998; Becker et al., J. Biol. Chem. 273: 11413–6, 1998; Hao et al., Biochemistry, 38: 4712–9, 1999; Dardel et al., J. Mol. Biol. 280: 501–13, 1998; O'Connell et al., J. Biomol. NMR, 13: 311–24, 1999), indicating similarities in active site geometry to metalloproteinases such as thermolysin and the metzincins.
The substrate specificity of PDF has been extensively studied (Ragusa et al., J. Mol. Biol. 289: 1445–57, 1999; Hu et al., Biochemistry 38: 643–50, 1999; Meinnel et al., Biochemistry, 38: 4287–95, 1999). These authors conclude that an unbranched hydrophobic chain is preferred at P1′, while a wide variety of P2′ substituents are acceptable and an aromatic amide substituent may be advantageous at the P3′ position. There have also been reports that small peptidic compounds containing an H-phosphonate (Hu et al., Bioorg. Med. Chem. Lett., 8: 2479–82, 1998) or thiol (Meinnel et al., Biochemistry, 38: 4287–95, 1999; Huntingdon et al., Biochemistry, 39: 4543–51, 2000; Wei et al, J. Combinatorial Chem., 2: 650–57, 2000) metal binding group are micromolar inhibitors of PDF. Peptide aldehydes such as calpeptin (N-Cbz-Leu-norleucinal) have also been shown to inhibit PDF (Durand et al., Arch. Biochem. Biophys., 367: 297–302, 1999). Recently, the naturally occurring hydroxamic acid antibiotic actinonin, for which the target of its antibacterial activity was previously unknown, was shown to be a potent inhibitor of polypeptide deformylase (WO 99/39704, and Chen et al, Biochemistry, 39: 1256–62, 2000). Examples of non-peptidic PDF inhibitors with carboxylic acid (Green et al., Arch. Biochem. Biophys. 375: 355–8, 2000; Jayasekera et al., ibid., 381: 313–6, 2000) or hydroxamic acid (Apfel et al., J. Med. Chem., 43: 2324–31, 2000) metal binding groups are also known.
It has been reported that PDF is present in eukaryotic parasites such as Plasmodium falciparum (Meinnel, Parasitology Today, 16: 165–8, 2000). Those authors also found evidence for the presence of PDF in other parasites of humans, such as the kinetoplastid protozoan parasites Trypanosoma brucei and Leishmania major. Based on these findings, it is anticipated that the hydroxamic acid and N-formyl hydroxylamine compounds with which this invention is concerned have antiprotozoal activity, and are useful in the treatment of malaria and other protozoal diseases.
Several patent applications describe antibacterial hydroxamic acid and N-formyl hydroxylamine agents whose activity has been attributed to inhibition of PDF. These publications include our copending International patent applications nos. WO 99/39704, WO 99/59568, WO 00/35440, WO 00/44373, WO 00/58294 and WO 00/61134, as well as WO 01/40198 (Aventis), WO 01/44179 (Versicor), WO 01/44178 (Versicor), and WO 01/38561 (Questcor).
Further, actinonin is a naturally occurring antibacterial agent having a hydroxamic acid group, and certain derivatives of actinonin are also known to have antibacterial activity. (see for example Bouboutou et al, Colloq. INSERM (1989) 174 (Forum Pept. 2nd, 1988), 341–4; Lelevre et. al. Pathol. Biol. (1989), 37(1), 43–46; Broughton et. al. J. Chem. Soc. Perkin Trans. 1 (1975) (9), 857–60. The antibacterial activity of actinonin has been shown to be due, at least in part, to inhibition of PDF (WO 99/39704 and other publications).